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Journal of Bacteriology, July 2002, p. 3630-3639, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3630-3639.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

David E. Nelson,,{dagger} Anindya S. Ghosh, Avery L. Paulson, and Kevin D. Young*

Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037

Received 10 December 2001/ Accepted 8 April 2002

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal ß-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, ND 58202-9037. Phone: (701) 777-2624. Fax: (701) 777-2054. E-mail: kyoung{at}medicine.nodak.edu.

{dagger} Present address: Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Indiana University School of Medicine, Indianapolis, IN 46202.

Journal of Bacteriology, July 2002, p. 3630-3639, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3630-3639.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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